Apc conjugated anti rat ig

PD-L1/EGFP cells were incubated with 4G12, Boch4G12, rat IgG2a isotype control (R35-95; BD Bioscience, San Jose, CA, USA), or bovine IgG1 isotype control (Bethyl Laboratories, Montgomery, TX, USA) in serial dilution (0.01–10 μg/ml). APC-conjugated anti-rat Ig (Beckman Coulter) or APC-conjugated anti-bovine IgG Fc (Jackson ImmunoResearch, West Grove, PA, USA) were used as secondary antibodies. Binding of the antibodies was detected by FACS Verse™ (BD Biosciences) and FCS Express 4 (De Novo Software, Glendale, CA, USA).

The binding of 4G12 to bovine PD-L1 on circulating IgM⁺ B cells was investigated to calculate the PD-L1 occupancy of 4G12 following inoculation. PBMCs isolated from #368 were pre-incubated with 10 μg/ml rat IgG2a isotype control (BD Bioscience) or 4G12, and then incubated with APC-conjugated anti-rat Ig (Beckman Coulter) at room temperature for 20 min. The same secondary antibody pre-incubated with IgG from rat serum (Sigma-Aldrich) at 37°C for 15 min was used as an unstained control. After washing twice, anti-IgM-PE/Cy7 (IL-A30; Bio-Rad) was reacted at room temperature for 20 min. The binding of the antibodies was then detected by flow cytometry, as described above. PD-L1 occupancy was estimated as the percentage of the in vivo binding (indicated as the number of cells positively stained by rat IgG2a isotype control) occurred at the total available binding sites (indicated as the number of cells positively stained by a saturated concentration of 4G12).